Human narcolepsy gene

ABSTRACT

The gene for hypocretin (orexin) receptor 1 (HCRTR1), which is associated with narcolepsy, is disclosed. Also described are methods of diagnosis of narcolepsy, pharmaceutical compositions comprising nucleic acids comprising the HCRTR1 gene, as well as methods of therapy of narcolepsy.

RELATED APPLICATIONS

[0001] This application is a Continuation of U.S. Ser. No. 09/479,128,filed Jan. 7, 2000, which is a Continuation-in-Part of U.S. Ser. No.09/379,083, filed Aug. 23, 1999, now abandoned, the entire teachings ofboth of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Narcolepsy, a disorder which affects approximately 1 in 2,000individuals, is characterized by daytime sleepiness, sleepfragmentation, and symptoms of abnormal rapid eye movement (REM) sleepthat include cataplexy (loss of muscle tone), sleep paralysis, andhypnagogic hallucinations (Aldrich, M. S., Neurology 42:34-43 (1992);Siegel, J. M.,Cell 98:409-412 (1999)). In humans, susceptibility tonarcolepsy has been associated with a specific human leukocyte antigen(HLA) alleles, including DQB1*0602 (Mignot, E., Neurology 50:S16-22(1998); Kadotani, H. et al., Genome Res. 8:427-434 (1998); Faraco, J. etal., J. Hered. 90:129-132 (1999)); however, attempts to verifynarcolepsy as an autoimmune disorder have failed (Mignot, E. et al.,Adv. Neuroimmunol. 5:23-37 (1995); Mignot, E., Curr. Opin. Pulm. Med.2:482-487 (1996)). In a canine model of narcolepsy, the disorder istransmitted as an autosomal recessive trait, canarc-1 (Foutz, A. S. etal, Sleep 1:413-421 91979); Baker, T. L. and Dement, W. C., BrainMechanisms of Sleep (D. J. McGinty et al., eds.s, New York: canine majorhistocompatibility complex has been excluded (Mignot, E. et al., Proc.Natl. Acad. Sci. USA 88:3475-3478 (1991)).

[0003] A mutation in the hypocretin (orexin) receptor 2 gene in canineshas been identified in narcolepsy (Lin, L. et al.,Cell 98:365-376(1999)); Hypocrexins/orexins (orexin-A and -B) are neuropeptidesassociated with regulation of food consumption (de Lecea, L., et al.,Proc. Natl. Acad. Sci. USA 95:322-327 (1998); Sakurai, T. et al., Cell92:573-585 (1998)) as well as other possible functions (Peyron, C. etal., J. Neurosci. 18:9996-10015 (1998)). Human cDNA of receptors fororexins have been cloned (Sakurai, T. et al.,Cell 92:573-585 (1998)),however, full human genes for the orexin receptors have not yet beenidentified.

[0004] Diagnosis of narcolepsy is difficult, as it is necessary todistinguish narcolepsy from other conditions such as chronic fatiguesyndrome or other sleep disorders (Ambrogetti, A. and Olson, L. C., Med.J. Aust. 160:426-429 (1994); Aldrich, M. S., Neurology 50:S2-7 (1998)).Methods of diagnosing narcolepsy based on specific criteria wouldfacilitate identification of the disease, reduce the time and expenseassociated with diagnosis, and expedite commencement of treatment.

SUMMARY OF THE INVENTION

[0005] As described herein, a full gene for the human hypocretin(orexin) receptor 1 (HCRTR1)has been identified. The sequence of theHCRTR1 gene as described herein is shown in FIG. 1 (SEQ ID NO:1).Accordingly, this invention pertains to an isolated nucleic acidmolecule containing the HCRTR1 gene. The invention also relates to DNAconstructs comprising the nucleic acid molecules described hereinoperatively linked to a regulatory sequence, and to recombinant hostcells, such as bacterial cells, fungal cells, plant cells, insect cellsand mammalian cells, comprising the nucleic acid molecules describedherein operatively linked to a regulatory sequence. The invention alsopertains to methods of diagnosing narcolepsy in an individual. Themethods include detecting the presence of a mutation in the HCRTR1 gene.The invention additionally pertains to pharmaceutical compositionscomprising the HCRTR1 nucleic acids of the invention. The inventionfurther pertains to methods of treating narcolepsy, by administeringHCRTR1 nucleic acids of the invention or compositions comprising theHCRTR1 nucleic acids. The methods of the invention allow the accuratediagnosis of narcolepsy and reduce the need for time-consuming andexpensive sleep laboratory assessments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] FIGS. 1A-1E depict the sequence of the human orexin receptor 1gene (SEQ ID NO:1) and the encoded receptor (SEQ ID NO:2).

[0007] The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings

DETAILED DESCRIPTION OF THE INVENTION

[0008] The present invention relates to a human hypocretin (orexin)receptor 1 (HCRTR1) gene, and the relationship of the gene tonarcolepsy. As described herein, Applicants have isolated the HCRTR1gene. The gene and its products are implicated in the pathogenesis ofnarcolepsy, as mutations in a closely related receptor, hypocretin(orexin) receptor 2, have been associated with the presence ofnarcolepsy in a well-established canine model of narcolepsy (Lin, L. etal.,Cell 98:365-376 (1999)).

Nucleic Acids of the Invention

[0009] Accordingly, the invention pertains to an isolated nucleic acidmolecule containing the human HCRTR1 gene. The term, “HCRTR1 gene,”refers to an isolated genomic nucleic acid molecule that encodes thehuman hypocretin (orexin) receptor 1. As used herein, the term, “genomicnucleic acid molecule” indicates that the nucleic acid molecule containsintrons and exons as are found in genomic DNA (i.e., not cDNA). Thenucleic acid molecules can be double-stranded or single-stranded; singlestranded nucleic acid molecules can be either the coding (sense) strandor the non-coding (antisense) strand. The nucleic acid molecule canadditionally contain a marker sequence, for example, a nucleotidesequence which encodes a polypeptide, to assist in isolation orpurification of the polypeptide. Such sequences include, but are notlimited to, those which encode a glutathione-S-transferase (GST) fusionprotein and those which encode a hemaglutinin A (HA) peptide marker frominfluenza. In a preferred embodiment, the nucleic acid molecule has thesequence shown in FIGS. 1A-1E (SEQ ID NO:1).

[0010] As used herein, an “isolated” or “substantially pure” gene ornucleic acid molecule is intended to mean a gene which is not flanked bynucleotide sequences which normally (in nature) flank the gene (as inother genomic sequences). Thus, an isolated gene can include a genewhich is synthesized chemically or by recombinant means. Thus,recombinant DNA contained in a vector are included in the definition of“isolated” as used herein. Also, isolated nucleotide sequences includerecombinant DNA molecules in heterologous host cells, as well aspartially or substantially purified DNA molecules in solution. Suchisolated nucleotide sequences are useful in the manufacture of theencoded protein, as probes for isolating homologous sequences (e.g.,from other mammalian species), for gene mapping (e.g., by in situhybridization with chromosomes), or for detecting expression of theHCRTR1 gene in tissue (e.g., human tissue), such as by Northern blotanalysis.

[0011] The present invention also encompasses variations of the nucleicacid sequences of the invention. Such variations can benaturally-occurring, such as in the case of allelic variation, ornon-naturally-occurring, such as those induced by various mutagens andmutagenic processes. Intended variations include, but are not limitedto, addition, deletion and substitution of one or more nucleotides whichcan result in conservative or non-conservative amino acid changes,including additions and deletions. Preferably, the nucleotide or aminoacid variations are silent or conserved; that is, they do not alter thecharacteristics or activity of the hypocretin (orexin) receptor 1.

[0012] Other alterations of the nucleic acid molecules of the inventioncan include, for example, labeling, methylation, internucleotidemodifications such as uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoamidates, carbamates), charged linkages (e.g.,phosphorothioates, phosphorodithioates), pendent moieties (e.g.,polypeptides), intercalators (e.g., acridine, psoralen), chelators,alkylators, and modified linkages (e.g., alpha anomeric nucleic acids).Also included are synthetic molecules that mimic nucleic acid moleculesin the ability to bind to a designated sequences via hydrogen bondingand other chemical interactions. Such molecules include, for example,those in which peptide linkages substitute for phosphate linkages in thebackbone of the molecule.

[0013] The invention also relates to fragments of the isolated nucleicacid molecules described herein. The term “fragment” is intended toencompass a portion of a nucleic acid sequence described herein which isfrom at least about 25 contiguous nucleotides to at least about 50contiguous nucleotides or longer in length. One or more introns can alsobe present. Such fragments are useful as probes, e.g., for diagnosticmethods, as described below and also as primers or probes. Particularlypreferred primers and probes selectively hybridize to a nucleic acidmolecule containing the HCRTR1 gene described herein.

[0014] The invention also pertains to nucleic acid molecules whichhybridize under high stringency hybridization conditions, such as forselective hybridization, to a nucleotide sequence described herein(e.g., nucleic acid molecules which specifically hybridize to a nucleicacid containing the HCRTR1 gene described herein). Hybridization probesare oligonucleotides which bind in a base-specific manner to acomplementary strand of nucleic acid. Suitable probes includepolypeptide nucleic acids, as described in (Nielsen et al., Science 254,1497-1500 (1991)).

[0015] Such nucleic acid molecules can be detected and/or isolated byspecific hybridization (e.g., under high stringency conditions).“Stringency conditions” for hybridization is a term of art which refersto the incubation and wash conditions, e.g., conditions of temperatureand buffer concentration, which permit hybridization of a particularnucleic acid to a second nucleic acid; the first nucleic acid may beperfectly (i.e., 100%) complementary to the second, or the first andsecond may share some degree of complementarity which is less thanperfect (e.g., 60%, 75%, 85%, 95%). For example, certain high stringencyconditions can be used which distinguish perfectly complementary nucleicacids from those of less complementarity.

[0016] “High stringency conditions”, “moderate stringency conditions”and “low stringency conditions” for nucleic acid hybridizations areexplained on pages 2.10.1-2.10.16 and pages 6.3.1-6 in Current Protocolsin Molecular Biology (Ausubel, F. M. et al., “Current Protocols inMolecular Biology”, John Wiley & Sons, (1998)) the teachings of whichare hereby incorporated by reference. The exact conditions whichdetermine the stringency of hybridization depend not only on ionicstrength (e.g., 0.2×SSC, 0.1×SSC), temperature (e.g., room temperature,42° C., 68° C.) and the concentration of destabilizing agents such asformamide or denaturing agents such as SDS, but also on factors such asthe length of the nucleic acid sequence, base composition, percentmismatch between hybridizing sequences and the frequency of occurrenceof subsets of that sequence within other non-identical sequences. Thus,high, moderate or low stringency conditions can be determinedempirically. By varying hybridization conditions from a level ofstringency at which no hybridization occurs to a level at whichhybridization is first observed, conditions which will allow a givensequence to hybridize (e.g., selectively) with the most similarsequences in the sample can be determined.

[0017] Exemplary conditions are described in Krause, M. H. and S. A.Aaronson, Methods in Enzymology, 200:546-556 (1991). Also, in, Ausubel,et al., “Current Protocols in Molecular Biology”, John Wiley & Sons,(1998), which describes the determination of washing conditions formoderate or low stringency conditions. Washing is the step in whichconditions are usually set so as to determine a minimum level ofcomplementarity of the hybrids. Generally, starting from the lowesttemperature at which only homologous hybridization occurs, each ° C. bywhich the final wash temperature is reduced (holding SSC concentrationconstant) allows an increase by 1% in the maximum extent of mismatchingamong the sequences that hybridize. Generally, doubling theconcentration of SSC results in an increase in T_(m) of ˜17° C. Usingthese guidelines, the washing temperature can be determined empiricallyfor high, moderate or low stringency, depending on the level of mismatchsought.

[0018] For example, a low stringency wash can comprise washing in asolution containing 0.2×SSC/0.1% SDS for 10 min at room temperature; amoderate stringency wash can comprise washing in a prewarmed solution(42° C.) solution containing 0.2×SSC/0.1% SDS for 15 min at 42° C.; anda high stringency wash can comprise washing in prewarmed (68° C.)solution containing 0.1×SSC/0.1% SDS for 15 min at 68° C. Furthermore,washes can be performed repeatedly or sequentially to obtain a desiredresult as known in the art. Equivalent conditions can be determined byvarying one or more of the parameters given as an example, as known inthe art, while maintaining a similar degree of identity or similaritybetween the target nucleic acid molecule and the primer or probe used.

[0019] Hybridizable nucleic acid molecules are useful as probes andprimers, e.g., for diagnostic applications, as described below. As usedherein, the term “primer” refers to a single-stranded oligonucleotidewhich acts as a point of initiation of template-directed DNA synthesisunder appropriate conditions (e.g., in the presence of four differentnucleoside triphosphates and an agent for polymerization, such as, DNAor RNA polymerase or reverse transcriptase) in an appropriate buffer andat a suitable temperature. The appropriate length of a primer depends onthe intended use of the primer, but typically ranges from 15 to 30nucleotides. Short primer molecules generally require coolertemperatures to form sufficiently stable hybrid complexes with thetemplate. A primer need not reflect the exact sequence of the template,but must be sufficiently complementary to hybridize with a template. Theterm “primer site” refers to the area of the target DNA to which aprimer hybridizes. The term “primer pair” refers to a set of primersincluding a 5′ (upstream) primer that hybridizes with the 5′ end of theDNA sequence to be amplified and a 3′ (downstream) primer thathybridizes with the complement of the 3′ end of the sequence to beamplified.

[0020] The invention also pertains to nucleotide sequences which have asubstantial identity with the nucleotide sequences described herein;particularly preferred are nucleotide sequences which have at leastabout 70%, and more preferably at least about 80% identity, and evenmore preferably at least about 90% identity, with nucleotide sequencesdescribed herein. Particularly preferred in this instance are nucleotidesequences encoding hypocretin (orexin) receptor 1.

[0021] To determine the percent identity of two nucleotide sequences,the sequences are aligned for optimal comparison purposes (e.g., gapscan be introduced in the sequence of a first nucleotide sequence). Thenucleotides at corresponding nucleotide positions are then compared.When a position in the first sequence is occupied by the same nucleotideas the corresponding position in the second sequence, then the moleculesare identical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=# of identical positions/total # ofpositions×100).

[0022] The determination of percent identity between two sequences canbe accomplished using a mathematical algorithm. A preferred,non-limiting example of a mathematical algorithm utilized for thecomparison of two sequences is the algorithm of Karlin et al. (Proc.Natl. Acad. Sci. USA, 90:5873-5877 (1993)). Such an algorithm isincorporated into the NBLAST program which can be used to identifysequences having the desired identity to nucleotide sequences of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al (Nucleic Acids Res,25:3389-3402 (1997)). When utilizing BLAST and Gapped BLAST programs,the default parameters of the respective programs (e.g., NBLAST) can beused. See http://www.ncbi.nlm.nih.gov. In one embodiment, parameters forsequence comparison can be set at W=12. Parameters can also be varied(e.g., W=5 or W=20). The value “W” determines how many continuousnucleotides must be identical for the program to identify two sequencesas containing regions of identity.

[0023] The invention also provides expression vectors containing anucleic acid comprising the HCRTR1 gene, operatively linked to at leastone regulatory sequence. Many such vectors are commercially available,and other suitable vectors can be readily prepared by the skilledartisan. “Operatively linked” is intended to mean that the nucleic acidsequence is linked to a regulatory sequence in a manner which allowsexpression of the nucleic acid sequence. Regulatory sequences areart-recognized and are selected to produce a hypocretin (orexin)receptor 1. Accordingly, the term “regulatory sequence” includespromoters, enhancers, and other expression control elements such asthose described in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990). For example,the native regulatory sequences or regulatory sequences native to thetransformed host cell can be employed. It should be understood that thedesign of the expression vector may depend on such factors as the choiceof the host cell to be transformed and/or the receptor desired to beexpressed. For instance, the gene of the present invention can beexpressed by ligating the gene into a vector suitable for expression ineither prokaryotic cells, eukaryotic cells or both (see, for example,Broach, et al., Experimental Manipulation of Gene Expression, ed. M.Inouye (Academic Press, 1983) p. 83; Molecular Cloning: A LaboratoryManual, 2nd Ed., ed. Sambrook et al. (Cold Spring Harbor LaboratoryPress, 1989) Chapters 16 and 17). Typically, expression constructs willcontain one or more selectable markers, including, but not limited to,the gene that encodes dihydrofolate reductase and the genes that conferresistance to neomycin, tetracycline, ampicillin, chloramphenicol,kanamycin and streptomycin resistance. Vectors can also include, forexample, an autonomously replicating sequence (ARS), expression controlsequences, ribosome-binding sites, RNA splice sites, polyadenylationsites, transcriptional terminator sequences, secretion signals and mRNAstabilizing sequences.

[0024] Prokaryotic and eukaryotic host cells transformed by thedescribed vectors are also provided by this invention. For instance,cells which can be transformed with the vectors of the present inventioninclude, but are not limited to, bacterial cells such as E. coli (e.g.,E. coli K12 strains), Streptomyces, Pseudomonas, Serratia marcescens andSalmonella typhimurium, insect cells (baculovirus), includingDrosophila, fungal cells, such as yeast cells, plant cells and mammaliancells, such as thymocytes, Chinese hamster ovary cells (CHO), and COScells. The host cells can be transformed by the described vectors byvarious methods (e.g., electroporation, transfection using calciumchloride, rubidium chloride, calcium phosphate, DEAE-dextran, or othersubstances; microprojectile bombardment; lipofection, infection wherethe vector is an infectious agent such as a retroviral genome, and othermethods), depending on the type of cellular host.

[0025] The nucleic acid molecules of the present invention can beproduced, for example, by replication in a suitable host cell, asdescribed above. Alternatively, the nucleic acid molecules can also beproduced by chemical synthesis.

[0026] The nucleotide sequences of the nucleic acid molecules describedherein (e.g., a nucleic acid molecule comprising SEQ ID NO:1) can beamplified by methods known in the art. For example, this can beaccomplished by e.g., PCR. See generally PCR Technology: Principles andApplications for DNA Amplification (ed. H. A. Erlich, Freeman Press, NY,N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (eds.Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al.,Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods andApplications 1, 17 (1991); PCR (eds. McPherson et al., IRL Press,Oxford); and U.S. Pat. No. 4,683,202.

[0027] Other suitable amplification methods include the ligase chainreaction (LCR) (see Wu and Wallace, Genomics 4, 560 (1989), Landegren etal., Science 241, 1077 (1988), transcription amplification (Kwoh et al.,Proc. Natl. Acad. Sci. USA 86, 1173 (1989)), and self-sustained sequencereplication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874(1990)) and nucleic acid based sequence amplification (NASBA). Thelatter two amplification methods involve isothermal reactions based onisothermal transcription, which produce both single stranded RNA (ssRNA)and double stranded DNA (dsDNA) as the amplification products in a ratioof about 30 or 100 to 1, respectively.

[0028] The amplified DNA can be radiolabeled and used as a probe forscreening a library or other suitable vector to identify homologousnucleotide sequences. Corresponding clones can be isolated, DNA can beobtained following in vivo excision, and the cloned insert can besequenced in either or both orientations by art recognized methods, toidentify the correct reading frame encoding a protein of the appropriatemolecular weight. For example, the direct analysis of the nucleotidesequence of homologous nucleic acid molecules of the present inventioncan be accomplished using either the dideoxy chain termination method orthe Maxam Gilbert method (see Sambrook et al., Molecular Cloning, ALaboratory Manual (2nd Ed., CSHP, New York 1989); Zyskind et al.,Recombinant DNA Laboratory Manual, (Acad. Press, 1988)). Using these orsimilar methods, the protein(s) and the DNA encoding the protein can beisolated, sequenced and further characterized.

Methods of Diagnosis

[0029] The nucleic acids and the proteins described above can be used todetect, in an individual, a mutation in the HCRTR1 gene that isassociated with narcolepsy. In one embodiment of the invention,diagnosis of narcolepsy is made by detecting a mutation in the HCRTR1gene. The mutation can be the insertion or deletion of a singlenucleotide, or of more than one nucleotide, resulting in a frame shiftmutation; the change of at least one nucleotide, resulting in a changein the encoded amino acid; the change of at least one nucleotide,resulting in the generation of a premature stop codon; the deletion ofseveral nucleotides, resulting in a deletion of one or more amino acidsencoded by the nucleotides; the insertion of one or several nucleotides,such as by unequal recombination or gene conversion, resulting in aninterruption of the coding sequence of the gene; duplication of all or apart of the gene; transposition of all or a part of the gene; orrearrangement of all or a part of the gene. More than one such mutationmay be present in a single gene. Such sequence changes cause a mutationin the receptor encoded by the HCRTR1 gene. For example, if the mutationis a frame shift mutation, the frame shift can result in a change in theencoded amino acids, and/or can result in the generation of a prematurestop codon, causing generation of a truncated receptor. Alternatively, amutation associated with narcolepsy can be a synonymous mutation in oneor more nucleotides (i.e., a mutation that does not result in a changein the receptor encoded by the HCRTR1 gene). Such a polymorphism mayalter splicing sites, affect the stability or transport of mRNA, orotherwise affect the transcription or translation of the gene. A HCRTR1gene that has any of the mutations described above is referred to hereinas a “mutant gene.” It is likely that a mutation in the HCRTR1 gene isassociated with narcolepsy in humans because of the association betweena mutation in the HCRTR1 gene and narcolepsy in dogs (Lin, L. etal.,Cell 98:365-376 (1999), the entire teachings of which areincorporated herein by reference). In a preferred embodiment, themutation in the HCRTR1 gene is to a deletion mutation, for example, adeletion that corresponds to the deletions found in the hypocretin(orexin) receptor 2 in narcoleptic dogs as described by Lin et al.,supra (e.g., a deletion of one or more exons, such as a deletion of thefourth exon, that can be caused by insertion of one or more nucleotidesupstream of the splice site of the exon, or a deletion of exon 6, thatcan be caused by a G to A transition in the splice junction consensussequence). In another preferred embodiment, the mutation in the HCRTR1gene is mutation that effects a “knockout” of the entire gene, such asdeletion of the first exon as described by Chemelli, R. M. et al, (Cell98:437-451 (1999), the entire teachings of which are incorporatedherein).

[0030] In a first method of diagnosing narcolepsy, hybridizationmethods, such as Southern analysis, are used (see Current Protocols inMolecular Biology, Ausubel, F. et al., eds., John Wiley & Sons,including all supplements through 1999). For example, a test sample ofgenomic DNA, RNA, or cDNA, is obtained from an individual suspected ofhaving (or carrying a defect for) narcolepsy (the “test individual”).The individual can be an adult, child, or fetus. The test sample can befrom any source which contains genomic DNA, such as a blood sample,sample of amniotic fluid, sample of cerebrospinal fluid, or tissuesample from skin, muscle, placenta, gastrointestinal tract or otherorgans. A test sample of DNA from fetal cells or tissue can be obtainedby appropriate methods, such as by amniocentesis or chorionic villussampling. The DNA, RNA, or cDNA sample is then examined to determinewhether a mutation in the HCRTR1 gene is present. The presence of themutation can be indicated by hybridization of the gene in the testsample to a nucleic acid probe. A “nucleic acid probe”, as used herein,can be a DNA probe or an RNA probe; the nucleic acid probe contains atleast one mutation in the HCRTR1 gene. The probe can be one of thenucleic acid molecules described above (e.g., the gene, a vectorcomprising the gene, etc.)

[0031] To diagnose narcolepsy by hybridization, a hybridization sampleis formed by contacting the test sample containing a HCRTR1 gene, withat least one nucleic acid probe. The hybridization sample is maintainedunder conditions which are sufficient to allow specific hybridization ofthe nucleic acid probe to the HCRTR1 gene. “Specific hybridization”, asused herein, indicates exact hybridization (e.g., with no mismatches).Specific hybridization can be performed under high stringency conditionsor moderate stringency conditions, for example, as described above. In aparticularly preferred embodiment, the hybridization conditions forspecific hybridization are high stringency.

[0032] Specific hybridization, if present, is then detected usingstandard methods. If specific hybridization occurs between the nucleicacid probe and the HCRTR1 gene in the test sample, then the HCRTR1 genehas the mutation that is present in the nucleic acid probe. More thanone nucleic acid probe can also be used concurrently in this method.Specific hybridization of any one of the nucleic acid probes isindicative of a mutation in the HCRTR1 gene, and is therefore diagnosticfor narcolepsy.

[0033] In another hybridization method, Northern analysis (see CurrentProtocols in Molecular Biology, Ausubel, F. et al., eds., John Wiley &Sons, supra) is used to identify the presence of a mutation associatedwith narcolepsy. For Northern analysis, a test sample of RNA is obtainedfrom the individual by appropriate means. Specific hybridization of anucleic acid probe, as described above, to RNA from the individual isindicative of a mutation in the HCRTR1 gene, and is therefore diagnosticfor narcolepsy

[0034] For representative examples of use of nucleic acid probes, see,for example, U.S. Pat. Nos. 5,288,611 and 4,851,330. Alternatively, apeptide nucleic acid (PNA) probe can be used instead of a nucleic acidprobe in the hybridization methods described above. PNA is a DNA mimichaving a peptide-like, inorganic backbone, such asN-(2-aminoethyl)glycine units, with an organic base (A, G, C, T or U)attached to the glycine nitrogen via a methylene carbonyl linker (see,for example, Nielsen, P. E. et al., Bioconjugate Chemistry, 1994, 5,American Chemical Society, p. 1 (1994). The PNA probe can be designed tospecifically hybridize to a gene having a polymorphism associated withautoimmune disease. Hybridization of the PNA probe to the HCRTR1 gene isdiagnostic for narcolepsy.

[0035] In another method of the invention, mutation analysis byrestriction digestion can be used to detect mutant genes, or genescontaining polymorphisms, if the mutation or polymorphism in the generesults in the creation or elimination of a restriction site. A testsample containing genomic DNA is obtained from the individual.Polymerase chain reaction (PCR) can be used to amplify the HCRTR1 gene(and, if necessary, the flanking sequences) in the test sample ofgenomic DNA from the test individual. RFLP analysis is conducted asdescribed (see Current Protocols in Molecular Biology, supra). Thedigestion pattern of the relevant DNA fragment indicates the presence orabsence of the mutation in the HCRTR1 gene, and therefore indicates thepresence or absence of narcolepsy.

[0036] Sequence analysis can also be used to detect specific mutationsin the HCRTR1 gene. A test sample of DNA is obtained from the testindividual. PCR can be used to amplify the gene, and/or its flankingsequences. The sequence of the HCRTR1 gene, or a fragment of the gene isdetermined, using standard methods. The sequence of the gene (or genefragment) is compared with the nucleic acid sequence of the gene, asdescribed above. The presence of a mutation in the HCRTR1 gene indicatesthat the individual has narcolepsy.

[0037] Allele-specific oligonucleotides can also be used to detect thepresence of a mutation in the HCRTR1 gene, through the use of dot-blothybridization of amplified proteins with allele-specific oligonucleotide(ASO) probes (see, for example, Saiki, R. et al., (1986), Nature(London) 324:163-166). An “allele-specific oligonucleotide” (alsoreferred to herein as an “allele-specific oligonucleotide probe”) is anoligonucleotide of approximately 10-50 base pairs, preferablyapproximately 15-30 base pairs, that specifically hybridizes to theHCRTR1 gene, and that contains a mutation associated with narcolepsy. Anallele-specific oligonucleotide probe that is specific for particularmutation in the HCRTR1 gene can be prepared, using standard methods (seeCurrent Protocols in Molecular Biology, supra). To identify mutations inthe gene that are associated with narcolepsy, a test sample of DNA isobtained from the individual. PCR can be used to amplify all or afragment of the HCRTR1 gene, and its flanking sequences. The DNAcontaining the amplified HCRTR1 gene (or fragment of the gene) isdot-blotted, using standard methods (see Current Protocols in MolecularBiology, supra), and the blot is contacted with the oligonucleotideprobe. The presence of specific hybridization of the probe to theamplified HCRTR1 gene is then detected. Specific hybridization of anallele-specific oligonucleotide probe to DNA from the individual isindicative of a mutation in the HCRTR1 gene, and is therefore indicativeof narcolepsy.

[0038] Other methods of nucleic acid analysis can be used to detectmutations in the HCRTR1 gene, for the diagnosis of narcolepsy.Representative methods include direct manual sequencing; automatedfluorescent sequencing; single-stranded conformation polymorphism assays(SSCA); clamped denaturing gel electrophoresis (CDGE) heteoduplexanalysis; chemical mismatch cleavage (CMC); RNase protection assays; useof proteins which recognize nucleotide mismatches, such as E. coli mutSprotein; allele-specific PCR, and other methods.

Pharmaceutical Compositions

[0039] The present invention also pertains to pharmaceuticalcompositions comprising nucleic acids described herein, particularlynucleic acids containing the HCRTR1 gene described herein. For instance,a nucleotide or nucleic acid construct (vector) comprising a nucleotideof the present invention can be formulated with a physiologicallyacceptable carrier or excipient to prepare a pharmaceutical composition.The carrier and composition can be sterile. The formulation should suitthe mode of administration.

[0040] Suitable pharmaceutically acceptable carriers include but are notlimited to water, salt solutions (e.g., NaCl), saline, buffered saline,alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzylalcohols, polyethylene glycols, gelatin, carbohydrates such as lactose,amylose or starch, dextrose, magnesium stearate, talc, silicic acid,viscous paraffin, perfume oil, fatty acid esters,hydroxymethylcellulose, polyvinyl pyro-lidone, etc., as well ascombinations thereof. The pharmaceutical preparations can, if desired,be mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, coloring, flavoring and/or aromatic substances andthe like which do not deleteriously react with the active compounds.

[0041] The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. The compositioncan be a liquid solution, suspension, emulsion, tablet, pill, capsule,sustained release formulation, or powder. The composition can beformulated as a suppository, with traditional binders and carriers suchas triglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,polyvinyl pyrol-lidone, sodium saccharine, cellulose, magnesiumcarbonate, etc.

[0042] Methods of introduction of these compositions include, but arenot limited to, intradermal, intramuscular, intraperitoneal,intraocular, intravenous, subcutaneous, oral and intranasal. Othersuitable methods of introduction can also include gene therapy (asdescribed below), rechargeable or biodegradable devices, particleacceleration devises (“gene guns”) and slow release polymeric devices.The pharmaceutical compositions of this invention can also beadministered as part of a combinatorial therapy with other agents.

[0043] The composition can be formulated in accordance with the routineprocedures as a pharmaceutical composition adapted for administration tohuman beings. For example, compositions for intravenous administrationtypically are solutions in sterile isotonic aqueous buffer. Wherenecessary, the composition may also include a solubilizing agent and alocal anesthetic to ease pain at the site of the injection. Generally,the ingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent. Where the compositionis to be administered by infusion, it can be dispensed with an infusionbottle containing sterile pharmaceutical grade water, saline ordextrose/water. Where the composition is administered by injection, anampoule of sterile water for injection or saline can be provided so thatthe ingredients may be mixed prior to administration.

[0044] For topical application, nonsprayable forms, viscous tosemi-solid or solid forms comprising a carrier compatible with topicalapplication and having a dynamic viscosity preferably greater thanwater, can be employed. Suitable formulations include but are notlimited to solutions, suspensions, emulsions, creams, ointments,powders, enemas, lotions, sols, liniments, salves, aerosols, etc., whichare, if desired, sterilized or mixed with auxiliary agents, e.g.,preservatives, stabilizers, wetting agents, buffers or salts forinfluencing osmotic pressure, etc. The agent may be incorporated into acosmetic formulation. For topical application, also suitable aresprayable aerosol preparations wherein the active ingredient, preferablyin combination with a solid or liquid inert carrier material, ispackaged in a squeeze bottle or in admixture with a pressurizedvolatile, normally gaseous propellant, e.g., pressurized air.

[0045] Agents described herein can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed with freeamino groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0046] The agents are administered in a therapeutically effectiveamount. The amount of agents which will be therapeutically effective inthe treatment of narcolepsy can be determined by standard clinicaltechniques. In addition, in vitro or in vivo assays may optionally beemployed to help identify optimal dosage ranges. The precise dose to beemployed in the formulation will also depend on the route ofadministration, and the seriousness of the disease or disorder, andshould be decided according to the judgment of a practitioner and eachpatient's circumstances. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

[0047] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use of sale for humanadministration. The pack or kit can be labeled with informationregarding mode of administration, sequence of drug administration (e.g.,separately, sequentially or concurrently), or the like. The pack or kitmay also include means for reminding the patient to take the therapy.The pack or kit can be a single unit dosage of the combination therapyor it can be a plurality of unit dosages. In particular, the agents canbe separated, mixed together in any combination, present in a singlevial or tablet. Agents assembled in a blister pack or other dispensingmeans is preferred. For the purpose of this invention, unit dosage isintended to mean a dosage that is dependent on the individualpharmacodynamics of each agent and administered in FDA approved dosagesin standard time courses.

Methods of Therapy

[0048] The present invention also pertains to methods of therapy fornarcolepsy, utilizing the pharmaceutical compositions comprising nucleicacids, as described herein. The therapy is designed toreplace/supplement activity of the hypocretin(orexin) receptor 1 in anindividual, such as by administering a nucleic acid comprising theHCRTR1 gene or a derivative or active fragment thereof. In oneembodiment of the invention, a nucleic acid of the invention is used inthe treatment of narcolepsy. The term, “treatment” as used herein,refers not only to ameliorating symptoms associated with the disease,but also preventing or delaying the onset of the disease, and alsolessening the severity or frequency of symptoms of the disease. In thisembodiment, a nucleic acid of the invention (e.g., the HCRTR1 gene (SEQID NO:1)) can be used, either alone or in a pharmaceutical compositionas described above. For example, the HCRTR1 gene, either by itself orincluded within a vector, can be introduced into cells (either in vitroor in vivo) such that the cells produce native HCRTR1 receptor. Ifnecessary, cells that have been transformed with the gene or can beintroduced (or reintroduced) into an individual affected with thedisease. Thus, cells which, in nature, lack native HCRTR1 expression andactivity, or have mutant HCRTR1 expression and activity, can beengineered to express HCRTR1 receptors (or, for example, an activefragment of the HCRTR1 receptor). In a preferred embodiment, nucleicacid comprising the HCRTR1 gene, can be introduced into an expressionvector, such as a viral vector, and the vector can be introduced intoappropriate cells which lack native HCRTR1 expression in an animal. Insuch methods, a cell population can be engineered to inducibly orconstitutively express active HCRTR1 receptor. Other gene transfersystems, including viral and nonviral transfer systems, can be used.Alternatively, nonviral gene transfer methods, such as calcium phosphatecoprecipitation, mechanical techniques (e.g., microinjection); membranefusion- mediated transfer via liposomes; or direct DNA uptake, can alsobe used.

[0049] The nucleic acids and/or vectors are administered in atherapeutically effective amount (i.e., an amount that is sufficient totreat the disease, such as by ameliorating symptoms associated with thedisease, preventing or delaying the onset of the disease, and/or alsolessening the severity or frequency of symptoms of the disease). Theamount which will be therapeutically effective in the treatment of aparticular disorder or condition will depend on the nature of thedisorder or condition, and can be determined by standard clinicaltechniques. In addition, in vitro or in vivo assays may optionally beemployed to help identify optimal dosage ranges. The precise dose to beemployed in the formulation will also depend on the route ofadministration, and the seriousness of the disease or disorder, andshould be decided according to the judgment of a practitioner and eachpatient's circumstances. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

[0050] The following Examples are offered for the purpose ofillustrating the present invention and are not to be construed to limitthe scope of this invention. The teachings of all references citedherein are hereby incorporated herein by reference.

EXAMPLES Example 1 Identification of the Human Narcolepsy Gene

[0051] A human BAC library (RPCI11 human male BAC library) was used.Seventeen primers, designed from the mRNA sequence of the HCRTR1receptor, were employed to identify clones of interest. They are setforth in Table 1. TABLE 1 Primers Used for Hybridization SEQ ID NumberName Primer Sequence NO: 1 HCRTR1-1-F TCAGGAAGTTTGAGGCTGAGA 3 2HCRTR1-1-R ATCCTAGGCTCTACAGAGGGA 4 3 HCRTR1-2-F GAAGATGAGTTTCTCCGCTATC 54 HCRTR1-2-R GATGAGGACCCACTCATACTG 6 5 HCRTR1-3-F ACATGAGGACAGTCACCAACTA7 6 HCRTR1-3-R CAGATAGCAGTCACCAGAACG 8 7 HCRTR1-4-FACATCACTGAGTCCTGGCTGT 9 8 HCRTR1-4-R ATGAAGCTGAGAGTTAGCACTG 10 9HCRTR1-5-F CTATTGTTCAAGAGCACAGCC 11 10 HCRTR1-5-R CATCACAGACTGAGAAGAGCC12 11 HCRTR1-6-R CTTCACTTCAGCCAGGAAGG 13 12 HCRTR1-7-FAATGTCCTTAAGAGGGTGTTCG 14 13 HCRTR1-7-R GAAGTTGTAGATGATGGGGTTG 15 14HCRTR1-8-F CACAAGTCCTTGTCCTTGCAG 16 15 HCRTR1-8-R CACCACATGCTCAGAGATTTTG17 16 HCRTR1-9-F CCTACCCCTCATGGAAAGAC 18 17 HCRTR1-9-RATCCAGAGTCACACAGGCAGA 19

Initial Study with Large Membranes

[0052] Four out of 5 membranes having the whole BAC library, containinga total of approximately 160,000 BAC clones representing anapproximately 10-fold coverage of the human genome, were used inhybridization studies with these primers. Hybridization was performedwith a pool of all 17 primers described in Table 1.

5′ End Labeling for Big Membranes

[0053] Oligonucleotides were labeled at the 5′ end before hybridization,using fresh [γ³²P]ATP (6000 Ci/mmole; 10 μCi/μl). Briefly, a labelingmixture was made of DNA (8 pmol/μl) (10.0 μl of the primer pool),10×buffer (12.0 μl), T4 PNK (10 u/μl) (6.0 μl), [γ ³²P]ATP (30.0 μl, or600 μCi), and water (62.0 μl) for a final volume of 120 μl. 20 μl oflabeling mixture was used per 10 ml rapid hybridization reaction.Incubation of the labeling mixture was for 2 hours at 37° C., followedby transfer to ice, spinning down, and mixing with the rapidhybridization solution. The membranes were prehybridized at 42° C.before the labeling mix was added. Sixty μl of the labeling mix wasadded to each of2 big bottles containing 2 membranes and 30 ml of rapidhybridization solution.

Hybridization and Washing

[0054] The membranes were hybridized at 42° C. overnight. Afterovernight, membranes were washed with 6×SSC, 0.1% SDS at roomtemperature; washed with 6×SSC, 0.1% SDS at 55° C. in a shakingwaterbath, repeated until the radioactivity of membranes was lower than6 k using 1×sensitivity; and washed with 6×SSC to remove the SDS. Thewashed membranes were put in a cassette for overnight exposure at −80°C. with a MR single emulsion film. Positive clones were identified andgridded on small membranes.

Study of Positive Clones with Small Membranes

[0055] After growing the positively-identified clones on several smallmembranes (to get several copies of membranes containing the sameclones), and washing the membranes, hybridization was performed usingpairs of primers, instead of a total pool of primers as before. Thetotal number of hybridizations was nine, using different primers againstidentical copies of membranes containing all positive clones from thefirst hybridization. The primer pairs are set forth in Table 2; primernumbers indicate the primers shown in Table 1. TABLE 2 Primer Pairs Usedfor Hybridization Reaction number Primers Used 1 1 and 2 2 3 and 4 3 5and 6 4 7 and 8 5 9 and 10 6 11 7 12 and 13 8 14 and 15 9 16 and 17

5′ End Labeling for Small Membranes

[0056] Oligonucleotides were labeled at the 5′ end before hybridization,using fresh [γ³²]ATP (5000 Ci/mmole; 10 μCi/μl). Briefly, a labelingmixture was made of DNA (8 pmol/μl) (1.5 μl), 10×buffer (2.0 μl), T4 PNK(10 u/μl) (1.0 μl), [γ³²]ATP (3.0 μl), and water (12.5 μl) for a finalvolume of 20 μl. Incubation of the labeling mixture was for 2.5 hours at37° C., followed by transfer to ice, spinning down, and mixing with therapid hybridization solution. Membranes were pre-wetted in 6×SSC, rolledin a pipette, and excess liquid drained prior to placing the membrane inthe tube. Fifty ml Falcon (polypropylene) tubes were used as containerfor the hybridization. The membranes were prehybridized at 42° C. before20 μl of labeling mix was added to each tube.

Hybridization and Washing

[0057] The membranes were hybridized at 42° C. overnight. Afterovernight, membranes were washed as described above. Four clones whichwere positive for primers designed using the 5′ and 3′ end of the mRNAwere identified. Clone 333N1 was used to characterize the gene.

Sequencing of Narcolepsy Gene in Clone 33N1

[0058] Shotgun sequencing, supplemented with sequencing of PCR productsamplified from 333N1, was used to obtain the gene sequence.

Shotgun Sequencing Preparation of DNA Samples

[0059] BAC DNA was isolated using the Plasmix kit from TALENT-VH BioLimited. Thirty μg of isolated DNA was fragmented by nebulization: anebulizer (IPI Medical Products, Inc., no. 4207) was modified byremoving the plastic cylinder drip ring, cutting off the outer rim ofthe cylinder, inverting it and placing it back into the nebulizer; thelarge hole in the top cover (where the mouth piece was attached) wassealed with a plastic stopper; the small hole was connected to a ¼ inchlength of Tycon tubing (connected to a compressed air source). A DNAsample was prepared containing 30 μg DNA, 10×TM buffer (200 μl), sterileglycerol (1 ml), and sterile dd water (q.s.) for a total volume of 2 ml.The DNA sample was nebulized in an ice-water bath for 2 minutes and 40seconds (pressure bar reading 0.5). The sample was then brieflycentrifuged at 2500 rpm to collect the DNA; the entire unit was placedin the rotor bucked of a table top centrifuge (Beckman GPR tabletopcentrifuge) fitted with pieces of Styrofoam to cushion the nebulizer.The sample was then distributed into four 1.5 ml microcentrifuge tubesand ethanol precipitated. The Dried DNA pellet was resuspended in 35 μlof 1×TM buffer prior to proceeding with fragment end-repair.

Fragment End Repair, Size Selection and Phosphorylation

[0060] The DNA was resuspended in 27 μl of 1×TM buffer. The followingmaterials were added: 10×kinase buffer (5 μl), 10 mM rATP (5 μl), 0.25mM dNTPs (7 μl), T4 polynucleotide kinase (1 μl (3 U/μl)), Klenow DNApolymerase (2 μl (5 U/μl)), T4 DNA polymerase (1 μl (3 U/μl)), for atotal volume of 48 μl. The mixture was incubated at 37° C. for 30minutes, and then 5 μl of agarose gel loading dye was added. The mixturewas then applied to separate wells of a 1% low melting temperatureagarose gel and electrophoresed for 30-60 minutes at 100-120 mA. The DNAwas then eluted from each sample lane, extracted from the agarose usingUltrafree-DA columns (Millipore) and then cleaned with Microcon-100columns (Amicon), precipitated in ethanol, and resuspended in 10 μl of10:0.1 TE buffer.

Ligation

[0061] EcoRV-linearized, CIAP-dephosphorylated Bluescript vector wasused as a cloning vector. The following reagents were combined in amicrocentrifuge tube, and incubated overnight at 4° C.: DNA fragments(100-1000 ng), cloning vector (2 μl (10 ng/μl)), 10×ligation buffer (1μl), T4 DNA ligase (NEB 202L) (1 μl (400 U/μl)), sterile dd water(q.s.), for a total of 10 μl.

Transformation of Ligated Products

[0062] The ligation products were diluted 1:5 with dd water and used totransform electrocompetent TOP 10F cells (Invitrogen) using GenePulserII (Biorad; voltage, 2.5 W, resistance 100 ohm). Transformants wereplated on LB plates with 50 μl of 4% X-GAL and 50 μl of 4% IPTG, andampicillin. Transformants were grown overnight at 37° C., white colonieswere picked, grown in a culture of 3 ml LB liquid media plus 200 μg/μlampicillin for 16-20 hours with shaking. DNA was isolated from theliquid cultures using Autogen 740 Automatic Plasmid Isolation System.

Cycle Sequencing of Isolated Plasmid DNA

[0063] Isolated plasmids were then sequenced using the M13 primers:M13-forward (SEQ ID NO:20) TGTAAAACGACGGCCAG; and M13-reverse (SEQ IDNO:21) CAGGAAACAGCTATGAC. For the sequencing reaction, 2.5 μl plasmidtemplate was mixed with 4 μl Big Dye Ready reaction mix (ABI), 1 μl of 8pM M13 primer, and 2.5 μl dd water. For cycle sequencing, 25 cycles of96° C. for 10 seconds, 50° C. for 5 seconds, and 60 ° C. for 4 minuteswere performed, followed by holding at 4° C. The cycle sequencingreaction products were cleaned by spinning through Sephadex G-50columns. The eluted cycle sequencing products were then dissolved in 3μl formamide/dye and 1.5 μl of sample was loaded on ABI 377 automatedsequencers. The data was analyzed using Phred and Phrap, and viewed inConsed viewer.

PCR Product Sequencing

[0064] In order to supplement the sequencing described above, PCRproducts of BAC 33N1 were also sequenced. Primers used for thehybridizations, as described above, were used to amplify regions of333N1 by long PCR using GeneAmpXL PCR kit (Perkin Elmer). The sequencefor HCRTR1-6F is as follows: TCTTTATTGTCACCTACCTGGC (SEQ ID NO: 22).

[0065] TABLE 3 PCR Product Sequencing Pairs Primers Used for CycleSequencing of Primer Pair Used for PCR PCR Product HCRTR1-1F/HCRTR1-4RHCRTR1-1F, 2F, 3F, 4F, 1R, 2R, 3R, 4R HCRTR1-1F/HCRTR1-6R HCRTR1-1F, 2F,3F, 5F, 6F, 1R, 2R, 3R HCRTR1-4F/HCRTR1-7R HCRTR1-4F, 5F, 6F, 7F, 4R,5R, 6R, 7R HCRTR1-6F/HCRTR1-8R HCRTR1-6F, 6R, 7R, 8R HCRTR1-7F/HCRTR1-9RHCRTR1-7F, 8F, 7R, 8R, 9R

[0066] The PCR products were prepared for cycle sequencing by incubationat 37° C. for 15 minutes and then at 87° C. for 15 minutes to destroythe enzymes. The PCR products were then subject to cycle sequencing asdescribed above, with the same procedures for sequencing gel runs andsequence analysis.

[0067] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the spirit and scope of theinvention as defined by the appended claims.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 22 <210> SEQ ID NO 1<211> LENGTH: 9785 <212> TYPE: DNA <213> ORGANISM: Homo Sapiens <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1270)...(1468) <221>NAME/KEY: CDS <222> LOCATION: (1609)...(1787) <221> NAME/KEY: CDS <222>LOCATION: (2920)...(3163) <221> NAME/KEY: CDS <222> LOCATION:(3554)...(3669) <221> NAME/KEY: CDS <222> LOCATION: (5599)...(5825)<221> NAME/KEY: CDS <222> LOCATION: (7074)...(7195) <221> NAME/KEY: CDS<222> LOCATION: (8867)...(9057) <400> SEQUENCE: 1 aaaggacccg cggcgagggatgggaggagc caagagtctc ggggggtaac ctgggtgctg 60 ggagactggc tcctcggccagcgctgctct cctctaggca ggctccgagt gccctcgctc 120 ccccgcgcct tcccggagccccgccagccc ccgaggtgca ggaaggtccc gcggacggag 180 cgggcctgcc ggcgttcagtggggtatgaa ggcgtcccct ccccttcccc aggggtctcc 240 agggatccgc aaccctccggcacctggccg gggtcgtcct agcccagccg ggggaaggag 300 gggctgagtg cgggaggaggggaggggcgg ggagctgggc tggctggatt tatgaatgga 360 gagcgacccg cgcgccggaacagcggctcc tggcggccgt cggggagcgt cgcggccctg 420 gggacccaaa ggggcctcttagggggcctg gatgctcccc ttgctcgcaa ggggtcgtca 480 gtccctccgc gcaccacccccactgtgtgt gtgttgtatg tgtgcgtgtc cgcgagtgcc 540 caccggaggg tctggcaggtatggcgggtg gggcttgggt ctctaacacc tcccttggcc 600 cgttttccca acccaaagttaaagcctctg aactggctca agaaatattt gcaatcggga 660 tggcttctcc tcccaaatctacggtgtttg gtgggttcga acagacctgg cttaagagct 720 ttgtgacctt gagcaagagactcaatctct ctgagcttcg gtctcatctg caaagcaggg 780 taccctaata atggtaaccggaaacgtccc cgaaactacc ttctcgtacc aggttcttgg 840 tgaagcactt ggcacgcatcggagctcatt actcctcatc gtggtcctgt aaggtatgta 900 gggctgtcac cccattagacagatggggaa accaaggctg aaagaggcca ggtaagctac 960 ccaaggcaac tggtgtggaattgggatgca acccaggtct gtcttcctcc accaatttca 1020 tgactgtgag aattaagagggaacttatac gcaaagcgcc tggcacaatc cctaatgttt 1080 ccttccttct ctcttttcccactccctcct ttccttcctc ccttcaggaa gtttgaggct 1140 gagacccgaa aagacctgggtgcaagcctc caggcaccct gaagggagtg ggctgagggc 1200 tggcccaagc tccctcctctccctctgtag agcctaggat gcccctctgc tgcagcggct 1260 cctgagctc atg gag ccctca gcc acc cca ggg gcc cag atg ggg gtc ccc 1311 Met Glu Pro Ser Ala ThrPro Gly Ala Gln Met Gly Val Pro 1 5 10 cct ggc agc aga gag ccg tcc cctgtg cct cca gac tat gaa gat gag 1359 Pro Gly Ser Arg Glu Pro Ser Pro ValPro Pro Asp Tyr Glu Asp Glu 15 20 25 30 ttt ctc cgc tat ctg tgg cgc gattat ctg tac cca aaa cag tat gag 1407 Phe Leu Arg Tyr Leu Trp Arg Asp TyrLeu Tyr Pro Lys Gln Tyr Glu 35 40 45 tgg gtc ctc atc gca gcc tat gtg gctgtg ttc gtc gtg gcc ctg gtg 1455 Trp Val Leu Ile Ala Ala Tyr Val Ala ValPhe Val Val Ala Leu Val 50 55 60 ggc aac acg ctg g gtaggtccag ggcttgcccggcagtgctgc cggctttccc 1508 Gly Asn Thr Leu 65 tggggattga agggggttgtgtgggaggag ggctcgctga ttaggcagaa ctaggatggg 1568 tgtggctctg ccaccagcttcacctcgctg caccctgcag tc tgc ctg gcc gtg 1622 Val Cys Leu Ala Val 70 tggcgg aac cac cac atg agg aca gtc acc aac tac ttc att gtc aac 1670 Trp ArgAsn His His Met Arg Thr Val Thr Asn Tyr Phe Ile Val Asn 75 80 85 ctg tccctg gct gac gtt ctg gtg act gct atc tgc ctg ccg gcc agc 1718 Leu Ser LeuAla Asp Val Leu Val Thr Ala Ile Cys Leu Pro Ala Ser 90 95 100 ctg ctggtg gac atc act gag tcc tgg ctg ttc ggc cat gcc ctc tgc 1766 Leu Leu ValAsp Ile Thr Glu Ser Trp Leu Phe Gly His Ala Leu Cys 105 110 115 aag gtcatc ccc tat cta cag gtgagctctg cccaggcacc cctcaccact 1817 Lys Val IlePro Tyr Leu Gln 120 125 ccttgtcacg cctgtaaaaa acccacggcc ttgcataggtctcagtgacc cccagacttg 1877 cctttcagac aggtcagtgg ctcatgaccc ctgaagtgtcatcctctgct gctagcaagg 1937 gcaagccacc agatcagaca ctcgaggaca cagacacaaccccacacact cacagagatc 1997 ccctcctggt cacagccaca gacatataca tagacacgtgtggacatgta tagtcacctc 2057 caggtacaca ggcacacagt caaggagaga ggcaacagcccacagtgaca catacacgac 2117 accctaggcc tgctccccaa tcccaaaggg gcagacgtgaggggcctgat ggaaacagcc 2177 gtctcctctc cctcctgcac tggccaggaa agaccccagtggtggaaacc aggatgtccg 2237 gatggggtta gtggggtgga aggaaggctt ctctcagtttgtatcctgtg atccacttcc 2297 tgcaccccag agggcagggg gcacccctag aggcaatgcccacacacctc tgacccagac 2357 tcatctctgc ctcccagaat gagggctttt tcctaacagcctggggaagg gatggcattc 2417 catggcagag ataaatgcct cttggatttc ccactattttgaggctcccc actcaactgg 2477 ttaactctgg tgaccctgag cataaagaca gatggatgagggaattctgt gcctcagttt 2537 cctcatctgt aacagggggg caagagcgct ttgtgggattgtcatgagga tgatgagaac 2597 agtgcccagc acatagtaag ttacgtaggt gcaagttattattcaccgga ggggtgcacc 2657 taccatgtgc caggtctaaa gctggacatt gtttgtacatgatttcactt attcgcacaa 2717 gaatcttgcc aggtagatgg tatattccca ttctgtagacgaggctcaga gagggtgagt 2777 gacttgccca catttacaca gccagtaagt ggtggagtcaggatttgcac tgccctgcac 2837 ctgccgtcag cctcctcact cacctactct cacatcgctgggtggccccc aaaatgaccg 2897 acgttgtgtc cccgtggggc ag gct gtg tcc gtg tcagtg gca gtg cta act 2949 Ala Val Ser Val Ser Val Ala Val Leu Thr 130 135ctc agc ttc atc gcc ctg gac cgc tgg tat gcc atc tgc cac cca cta 2997 LeuSer Phe Ile Ala Leu Asp Arg Trp Tyr Ala Ile Cys His Pro Leu 140 145 150ttg ttc aag agc aca gcc cgg cgg gcc cgt ggc tcc atc ctg ggc atc 3045 LeuPhe Lys Ser Thr Ala Arg Arg Ala Arg Gly Ser Ile Leu Gly Ile 155 160 165tgg gct gtg tcg ctg gcc atc atg gtg ccc cag gct gca gtc atg gaa 3093 TrpAla Val Ser Leu Ala Ile Met Val Pro Gln Ala Ala Val Met Glu 170 175 180tgc agc agt gtg ctg cct gag cta gcc aac cgc aca cgg ctc ttc tca 3141 CysSer Ser Val Leu Pro Glu Leu Ala Asn Arg Thr Arg Leu Phe Ser 185 190 195200 gtc tgt gat gaa cgc tgg gca g gtaatggtgg aagcctcaag caggcatccc 3193Val Cys Asp Glu Arg Trp Ala 205 ctcaggtggg cactttggga gcacgtacccctaggacagg catctagcag ggtcccttcc 3253 aaagtgggaa atcccagagc aggtatttccctaggggaca ccctagactg gctcctacca 3313 gggatactcc cagggtgggt gcctcccctcatgtagacat ctgctctagt gtagatgtcc 3373 ttccaggagg gacaacccaa gttggacaactccagggtct ctgtctgtca tggtggctgt 3433 atggggtcca gctgctccta ggccttgctttggccgtagt caggacaggg tggcattgct 3493 aaccagggca gggtggggct cacggattgggcctgactct gcatctcttg acccctgcag 3553 at gac ctc tat ccc aag atc tac cacagt tgc ttc ttt att gtc acc 3600 Asp Asp Leu Tyr Pro Lys Ile Tyr His SerCys Phe Phe Ile Val Thr 210 215 220 tac ctg gcc cca ctg ggc ctc atg gccatg gcc tat ttc cag ata ttc 3648 Tyr Leu Ala Pro Leu Gly Leu Met Ala MetAla Tyr Phe Gln Ile Phe 225 230 235 cgc aag ctc tgg ggc cgc caggtgaggccca ctctgggcag gggctaggcc 3699 Arg Lys Leu Trp Gly Arg Gln 240245 agtcactgtg tgggctgggg gtgggagggc tactggtcta actgagtagg cagtcctctg3759 ccatcagcac atgccatctt ggctgcaacc aaagagaggg gaagcccaga gacacgtcaa3819 actcaaggcc aaaagcacca gtggctaccc tggaatggaa tagtaacacg tccttctatt3879 agtggttggc gtttattgaa gtatccactc ccagataatc ttgcatcctc ttagccacca3939 tatattaccc acattaaata tatgagaaaa ccgagaccca gaagatcaac ataacttccc3999 ccaaaccact cagctagtga gtagatcagg aactaaagcc cagatctgtg agctcccact4059 gctcagttta gtaccactgc aacaataata atagcaactc cgtggtgctt gccaaattag4119 gcactttgca tccaatgtct taacaactat ctaacaaaag aagcaacatt acccacgtca4179 caaatgccaa ataagggcaa ccaacttgcc agattcaaca gcagcagagc cttctggttc4239 cagggcctgt cttctttcct gcattacaga ctgacccacg gtgggtttct taggtttttg4299 gggggcaggg gtggtcagag gcccttggcc tagcgagtgg gagtcctgga ttggcgtctg4359 ggcggtgaga aaaggcaggc cagaacatga ccaggctcag gaagggactc tcacacttgg4419 ggatgtcacc tacattccac aggaagtact ggcttgcacc caggccatgc cgggcagcgg4479 atggggacac ggactggctg tgacccaggt cctgccttgg aggagcaccc agtccagtag4539 gacccttcct gactggccag ccctgtagtc caccaacact catcatctgc tccccacaga4599 ccccccagcc aagcaggaca caggcacgat cctcctcatt tgacagatat gaaagcaagg4659 cttagaaagg aaaatgaggt ggctaaggtc acatagctca cgaatggctg agctggctct4719 agacccgcgt ttccagaact ccagccccat gcccctctgt ggtgggtgat ttgagtgtcc4779 ggtggcagga gaggcttctc caggagccca gaaccacccc aggcttatgg gcactggccc4839 aggccattcg atgctgccca cctgctcacc ccttgcccag gcctcctcat agtctggtat4899 gatccagggg aggcacaact cacccccacc cctaccctca aagatagtgt tggagattta4959 gggaggatgg atgggcagtt gacaggatgt ggcctggggt cttgtcaagg ttccccacct5019 ctttgagtct tagttgcctc atctatacct aaggaccaat aatatctttc cacaaggcgt5079 gttgtagagg gtttcacaaa gagctaatgg aaaatgaaag tctaggctgg gcgcagtggc5139 tcacacctgt attcccagca ctttgggagg ctgaggcagg cggatcacct aaggtcagga5199 gttcaagacc agcctggcca acgtggtgaa accccatctc tactaaaaat acaaaactta5259 gcccggtgtg gtggcgcaca cctgtaatcc cagctactcg ggaggcgaga ttgaagagag5319 ccaagattgc accattgcac tccagcttag gtgacaagag tgaaatgcca tctcaaaaaa5379 aaaaaaaaag aaaagaaaag aaaatgaaag tctatcgttc actctcaagt ccagagtgtt5439 agtctatcat aaacattaga ttccttcctc ttgcaagggt tttatccttt tgcccatctc5499 caccctgccc ggggtccagc ctggagtagg ccccacaaaa ggcaaccacc ctcccaaggt5559 gctgtaccca ccactgctgt ctctatgtgt gctggacag atc ccc ggc acc acc 5613Ile Pro Gly Thr Thr 250 tca gca ctg gtg cgg aac tgg aag cgc ccc tca gaccag ctg ggg gac 5661 Ser Ala Leu Val Arg Asn Trp Lys Arg Pro Ser Asp GlnLeu Gly Asp 255 260 265 ctg gag cag ggc ctg agt gga gag ccc cag ccc cgggcc cgc gcc ttc 5709 Leu Glu Gln Gly Leu Ser Gly Glu Pro Gln Pro Arg AlaArg Ala Phe 270 275 280 ctg gct gaa gtg aag cag atg cgt gca cgg agg aagaca gcc aag atg 5757 Leu Ala Glu Val Lys Gln Met Arg Ala Arg Arg Lys ThrAla Lys Met 285 290 295 ctg atg gtg gtg ctg ctg gtc ttc gcc ctc tgc tacctg ccc atc agc 5805 Leu Met Val Val Leu Leu Val Phe Ala Leu Cys Tyr LeuPro Ile Ser 300 305 310 315 gtc ctc aat gtc ctt aag ag gtgagagcacggggtatggt tggggtgggg 5855 Val Leu Asn Val Leu Lys Arg 320 agaagtttgaggttggggaa ggagctctcc ttgcttggga gaaagacctg gctccacccc 5915 ttctccactatgtgatcttg ggcaggccat ttctcttctc tgagcctcca tctcctaggg 5975 ctatcgtgaaaattcacgca ttcattcact taatcatcac attttagggg gctggaaata 6035 caatgaacaagtgcataaga cagacaaagt ccctgccttc atggaggctg cattctagca 6095 ggagagaagggaagtaaata gaagaatcaa tgtatattat aatgtcaggc agtgataact 6155 gctgggaagaaaaataaaat aggacagaga gtgacaatga taagggttgg tgggtttttg 6215 cttttgctttagatacaatg gtttaaaaaa agcagggggc cgggtgcagt ggctcacatc 6275 tgtaatcccaacacgttggg aggccaagga gggaggatcg cttgaggcca ggagttcaag 6335 atcagcccgggcaacataat gagacttcgt ctctactaaa attcaaaaaa ttagccagcc 6395 atggtggcatgtgcctgtag ttctagctac acagactgag gtggaagaat agcttgagcc 6455 caggaggttgaggctgcagc gaaccatgat tgcaccactg cactccagcc tgggtgacac 6515 agctgtctcaaaaaaaaaaa aaaaaaaaaa aaagcctttc caaggaaatg acatttgagc 6575 agagacttgaaggaagtgag agagctaacc atgcacgtgt ctgtagggac agccaaagag 6635 ggtcgcagggcgctggggag agaatgcagg ctattggaca gaagacagtt tcactttgag 6695 attgtgcttggccacttcct ggttgtgtga tcttcggcat gtcactttac ttctctgagc 6755 ctcagtttccttaatggaaa aatggatgat gtctatgatt catcatgttg ctgtgaggat 6815 ggatgagaaagtggatggga agccccaggg gatccgatgg ccaggaggct agagatgccc 6875 atcacggtgcttgataccct ccatgcttga gaaccccaaa ccctggccaa gacctcaggt 6935 acagaaggccaggaaacgtg gacagaagtg ggcagtagga actcttgcac tttacagctc 6995 aggttctgtgagcagcactc ccccagtaca tgcatacgca gctaccccat ttctgacgct 7055 cctccaccctgggcctag g gtg ttc ggg atg ttc cgc caa gcc agt gac cgc 7107 Val Phe GlyMet Phe Arg Gln Ala Ser Asp Arg 325 330 gaa gct gtc tac gcc tgc ttc accttc tcc cac tgg ctg gtg tac gcc 7155 Glu Ala Val Tyr Ala Cys Phe Thr PheSer His Trp Leu Val Tyr Ala 335 340 345 aac agc gct gcc aac ccc atc atctac aac ttc ctc agt g gtgagcaggc 7205 Asn Ser Ala Ala Asn Pro Ile IleTyr Asn Phe Leu Ser 350 355 360 tggggatgca aaatgactga gggtggccaacagtccacat gacaagtctc cccatcccca 7265 agccagggcc caaataaagg atggtgggtgaggatgtacc tgctgtgggc acagtgatcc 7325 tgctctggga ggacccaccc caagcggccctggcctgagt gggagacggg ccacactccc 7385 tacagtggct ggcacccagg atccagttttgcagattctg cagaccagtg agtgagtgga 7445 agggcagggg ctaggccagc tcacccccaactcccaccct gggtgcaggc acagcaagac 7505 ctccaatcag ctcaggcaga ggagtccatcctccccggag ggagtcagac ctgtgggagg 7565 agggccctgg agcccctgcc cgaggaaggattgcacagtc caggtgtcag ggctaaagta 7625 gggtcactct gagagacaag ccaggcccagggaagggctt cgccggctca gctagacaca 7685 ctggcagagt gaccggaatc tcaggggttgtcccctctgg aagtcttcct cccctgccac 7745 ccccactccc actccaggcc tctcctctctgctgtcccac agtgcccacc ccctccctct 7805 acctcccagt ctcagggtgg taatggctctgaggctgagc tcagcagaag tctgactcac 7865 cagccctctg actttgggaa tagacttctaaagaacaggt ccagatgact gttgaagcct 7925 ggacagaaat aatctttgag gaactattaaaaggttaaag aaaggatcag gagtcaatag 7985 tataaccctc attgagactc aagaattactcaacaaggct ggctgcgggt ttccaggtca 8045 gaaaagagaa tagatgatga gctgtgtggggaggggaggg cagacagact tactgacaca 8105 tatgcctttg tttggcctat gtttactgagcacctactat gtgcttgacc ctgtgctggg 8165 caccagagag gctggcagcc taatgacacatgatcaaagg ggcttcagcc tgacaaaatc 8225 tgtttccctg gtatacttgg gctgaataatgtggtgtggt ggtccctcct tccctcctcc 8285 cccttgagaa gggctttgga attagaattgggttcagctt ctggctgggt ggacttgggc 8345 aagccactgt acctctgtgc atctcatctgtgaagtgagg ataaaggact ccagcctttc 8405 agggtgctgg gatgctctgg cggacagaggctgaggcgcc cagcacagcg tgactgccaa 8465 atgcaaaagg gctgctgctg ccgtcattttcatcatcaaa gggcagagag gacacaagcc 8525 tcgcaacaga tagtgacccc cacgtacacaccaaggagag cagaggtgac ctgaggcccc 8585 cgagccagac accacgtttt gagtcagcctccgagccaga gcacagtcaa ggaatcagat 8645 ggcaattgcg tctctccttg ggaacccgctccagggcttc tgtcctctct ctctggcggt 8705 gccgaggttg cctcagggct ctccctcccagctctatccc tccctccctc cccgccccct 8765 cataggcagc ttggctggag ctgcgtgggtgtccctgggc tcaaggcccc ttcctgctgc 8825 atctgtctcc ttatggctgt gtcttttgtctcccaaccaa g gc aaa ttc cgg gag 8880 Gly Lys Phe Arg Glu 365 cag ttt aaggct gcc ttc tcc tgc tgc ctg cct ggc ctg ggt ccc tgc 8928 Gln Phe Lys AlaAla Phe Ser Cys Cys Leu Pro Gly Leu Gly Pro Cys 370 375 380 ggc tct ctgaag gcc cct agt ccc cgc tcc tct gcc agc cac aag tcc 8976 Gly Ser Leu LysAla Pro Ser Pro Arg Ser Ser Ala Ser His Lys Ser 385 390 395 ttg tcc ttgcag agc cga tgc tcc atc tcc aaa atc tct gag cat gtg 9024 Leu Ser Leu GlnSer Arg Cys Ser Ile Ser Lys Ile Ser Glu His Val 400 405 410 415 gtg ctcacc agc gtc acc aca gtg ctg ccc tga gcgagggctg ccctggaggc 9077 Val LeuThr Ser Val Thr Thr Val Leu Pro * 420 425 tccggctcgg gggatctgcccctacccctc atggaaagac agctggatgt ggtgaaaggc 9137 tgtggcttca gtcctgggtttctgcctgtg tgactctgga taagtcactt cctctgtctg 9197 agcttgtgtc ccctaagcagggttgatgtg aggattaagc atgctgaagc aagtggaaag 9257 ctccttgtaa actgtgaagtgttgtggaca tgattattgt tgtacttctc tcatttggcc 9317 ataccccaca gtataatctgtccccatcct ccttccagag cttggtcatc ctcctaaaga 9377 cccctttcct acccaattacaggccttccc tggagtctgc tctaaaggtc ccaacaggca 9437 tttccatctt gttccatggctccctgaagc ccagggctgc acttggccag ctgttctgat 9497 gcctgtgtga actaatctgggcccagcctt tctccagcgg gccacgagca cagccccacc 9557 ctaaccaggt gccaagggcacacaccacag acccgacctt gttggctttg tggtgtgata 9617 aaacactctc catggccacttggcagagag gccagcagcc cgaagcaact gtaattaaaa 9677 gcctggcact gaatgttccctttccttgtc attgcacaaa atctgtgctg cttaggttag 9737 gagcagaaga aggtggggaagctgggggga gggaagacaa gaaggcac 9785 <210> SEQ ID NO 2 <211> LENGTH: 425<212> TYPE: PRT <213> ORGANISM: Homo Sapiens <400> SEQUENCE: 2 Met GluPro Ser Ala Thr Pro Gly Ala Gln Met Gly Val Pro Pro Gly 1 5 10 15 SerArg Glu Pro Ser Pro Val Pro Pro Asp Tyr Glu Asp Glu Phe Leu 20 25 30 ArgTyr Leu Trp Arg Asp Tyr Leu Tyr Pro Lys Gln Tyr Glu Trp Val 35 40 45 LeuIle Ala Ala Tyr Val Ala Val Phe Val Val Ala Leu Val Gly Asn 50 55 60 ThrLeu Val Cys Leu Ala Val Trp Arg Asn His His Met Arg Thr Val 65 70 75 80Thr Asn Tyr Phe Ile Val Asn Leu Ser Leu Ala Asp Val Leu Val Thr 85 90 95Ala Ile Cys Leu Pro Ala Ser Leu Leu Val Asp Ile Thr Glu Ser Trp 100 105110 Leu Phe Gly His Ala Leu Cys Lys Val Ile Pro Tyr Leu Gln Ala Val 115120 125 Ser Val Ser Val Ala Val Leu Thr Leu Ser Phe Ile Ala Leu Asp Arg130 135 140 Trp Tyr Ala Ile Cys His Pro Leu Leu Phe Lys Ser Thr Ala ArgArg 145 150 155 160 Ala Arg Gly Ser Ile Leu Gly Ile Trp Ala Val Ser LeuAla Ile Met 165 170 175 Val Pro Gln Ala Ala Val Met Glu Cys Ser Ser ValLeu Pro Glu Leu 180 185 190 Ala Asn Arg Thr Arg Leu Phe Ser Val Cys AspGlu Arg Trp Ala Asp 195 200 205 Asp Leu Tyr Pro Lys Ile Tyr His Ser CysPhe Phe Ile Val Thr Tyr 210 215 220 Leu Ala Pro Leu Gly Leu Met Ala MetAla Tyr Phe Gln Ile Phe Arg 225 230 235 240 Lys Leu Trp Gly Arg Gln IlePro Gly Thr Thr Ser Ala Leu Val Arg 245 250 255 Asn Trp Lys Arg Pro SerAsp Gln Leu Gly Asp Leu Glu Gln Gly Leu 260 265 270 Ser Gly Glu Pro GlnPro Arg Ala Arg Ala Phe Leu Ala Glu Val Lys 275 280 285 Gln Met Arg AlaArg Arg Lys Thr Ala Lys Met Leu Met Val Val Leu 290 295 300 Leu Val PheAla Leu Cys Tyr Leu Pro Ile Ser Val Leu Asn Val Leu 305 310 315 320 LysArg Val Phe Gly Met Phe Arg Gln Ala Ser Asp Arg Glu Ala Val 325 330 335Tyr Ala Cys Phe Thr Phe Ser His Trp Leu Val Tyr Ala Asn Ser Ala 340 345350 Ala Asn Pro Ile Ile Tyr Asn Phe Leu Ser Gly Lys Phe Arg Glu Gln 355360 365 Phe Lys Ala Ala Phe Ser Cys Cys Leu Pro Gly Leu Gly Pro Cys Gly370 375 380 Ser Leu Lys Ala Pro Ser Pro Arg Ser Ser Ala Ser His Lys SerLeu 385 390 395 400 Ser Leu Gln Ser Arg Cys Ser Ile Ser Lys Ile Ser GluHis Val Val 405 410 415 Leu Thr Ser Val Thr Thr Val Leu Pro 420 425<210> SEQ ID NO 3 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: nucleic acidprimers based on human mRNA sequence <400> SEQUENCE: 3 tcaggaagtttgaggctgag a 21 <210> SEQ ID NO 4 <211> LENGTH: 21 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:nucleic acid primers based on human mRNA sequence <400> SEQUENCE: 4atcctaggct ctacagaggg a 21 <210> SEQ ID NO 5 <211> LENGTH: 22 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: nucleic acid primers based on human mRNA sequence <400>SEQUENCE: 5 gaagatgagt ttctccgcta tc 22 <210> SEQ ID NO 6 <211> LENGTH:21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: nucleic acid primers based on human mRNAsequence <400> SEQUENCE: 6 gatgaggacc cactcatact g 21 <210> SEQ ID NO 7<211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: nucleic acid primers based onhuman mRNA sequence <400> SEQUENCE: 7 acatgaggac agtcaccaac ta 22 <210>SEQ ID NO 8 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: nucleic acid primersbased on human mRNA sequence <400> SEQUENCE: 8 cagatagcag tcaccagaac g21 <210> SEQ ID NO 9 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: nucleic acidprimers based on human mRNA sequence <400> SEQUENCE: 9 acatcactgagtcctggctg t 21 <210> SEQ ID NO 10 <211> LENGTH: 22 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: nucleic acid primers based on human mRNA sequence <400>SEQUENCE: 10 atgaagctga gagttagcac tg 22 <210> SEQ ID NO 11 <211>LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: nucleic acid primers based on humanmRNA sequence <400> SEQUENCE: 11 ctattgttca agagcacagc c 21 <210> SEQ IDNO 12 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: nucleic acid primersbased on human mRNA sequence <400> SEQUENCE: 12 catcacagac tgagaagagc c21 <210> SEQ ID NO 13 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: nucleic acidprimers based on human mRNA sequence <400> SEQUENCE: 13 cttcacttcagccaggaagg 20 <210> SEQ ID NO 14 <211> LENGTH: 22 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:nucleic acid primers based on human mRNA sequence <400> SEQUENCE: 14aatgtcctta agagggtgtt cg 22 <210> SEQ ID NO 15 <211> LENGTH: 22 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: nucleic acid primers based on human mRNA sequence <400>SEQUENCE: 15 gaagttgtag atgatggggt tg 22 <210> SEQ ID NO 16 <211>LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: nucleic acid primers based on humanmRNA sequence <400> SEQUENCE: 16 cacaagtcct tgtccttgca g 21 <210> SEQ IDNO 17 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: nucleic acid primersbased on human mRNA sequence <400> SEQUENCE: 17 caccacatgc tcagagattt tg22 <210> SEQ ID NO 18 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: nucleic acidprimers based on human mRNA sequence <400> SEQUENCE: 18 cctacccctcatggaaagac 20 <210> SEQ ID NO 19 <211> LENGTH: 21 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:nucleic acid primers based on human mRNA sequence <400> SEQUENCE: 19atccagagtc acacaggcag a 21 <210> SEQ ID NO 20 <211> LENGTH: 17 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: nucleic acid primers <400> SEQUENCE: 20 tgtaaaacga cggccag17 <210> SEQ ID NO 21 <211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: nucleic acidprimers <400> SEQUENCE: 21 caggaaacag ctatgac 17 <210> SEQ ID NO 22<211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: nucleic acid primers <400>SEQUENCE: 22 tctttattgt cacctacctg gc 22

What is claimed is:
 1. A method of diagnosing narcolepsy in anindividual, comprising detecting a mutation in the gene encodinghypocretin (orexin) receptor 1, wherein the presence of the mutation inthe gene is indicative of narcolepsy.
 2. A pharmaceutical compositioncomprising a nucleic acid comprising the isolated nucleic acid moleculeof SEQ ID NO:1.
 3. A method of treating narcolepsy in an individual,comprising administering to the individual an isolated nucleic acid ofSEQ ID NO:1 in a therapeutically effective amount.